2 Electron Transfer Through DNA
2.1.1 Aim of Chapter 2
Whereas hopping of electrons through DNA is now a generally accepted model, it remained to address the conflicting data about the sequence dependence, as well as the direction of electron transfer and the role of the electron acceptors used in this process. In summary, investigations of the electron transfer process using DNA modified with either arylamines,[33,215,219] pyrenes or phenothiazines[218,234] as electron donors and BrdU as electron acceptor pointed out that G:C bps, in contrast to A:T bps, reduce the efficiency of the excess electron transfer through the duplex. Studies with a flavin as electron donor and a T=T dimer as electron acceptor, in contrast, showed in contrast no sequence dependence.[217] Giese and co-workers ascribed the divergent results to the rate of the T=T dimer cycloreversion, which might be too low to fully report the EET process.[230] However new pulse radiolysis studies, carried out at the nucleoside level in solution, suggest a very fast and irreversible dimer opening close to the diffusion range (> 107-8 s-1).[233] This value would be in contrast to the observation described in the previous paragraph and reported in Giese’s work. These and other open questions are the subject of the Results and Discussion of Chapter 2 as summarised as follows.
Different electron acceptors (i.e. BrdU) were planned be used in combination with the widely employed flavin donor, allowing a systematic study of the function of electron traps in the EET.
A sequence dependence study of the EET was planned to be undertaken in order to disclose the capacity of the flavin-containing system to fully report the process.
The single electron donor developed by Giese was intended to be used in combination with the T=T dimer and BrdU for a more in-depth investigation of the dimer splitting process.
A parallel study was planned to be achieved, using the flavin in order to compare the two electron donors and evaluate the role of their redox potentials in the EET process.
A new model for DNA-based nano-wire applications will be described and tested. The first two points have been achieved in collaboration with Sascha Breeger (from the Carell group).
Before the results are presented, a short introduction about the electron acceptors features used in this work is necessary. The driving forces for the selection of such molecules as electron traps in the EET process are here described.
Electron acceptors: BrdU
Replacement of thymidine in DNA by BrdU has long been known to enhance photosensitivity with respect to DNA-protein-mediated photo-cross-linking,[235] single- and double-strand breaks[236] and creation of alkali-labile sites.[237] In addition, the effect of a BrdU substitution in DNA is well known for a long time.[238] This residue is nearly isosteric to thymidine and typically pairs with adenosine when incorporated into DNA, although occasional mispairing with guanine contributes to its mutagenic effect.[239,240] The nature of the photolability of BrdU was intensively studied and is described now as a process that involves three main steps: acception of an electron, elimination of a bromine anion and generation of an intermediate uridinyl radical. The fate of this radical depends on specific reaction conditions, the local structural and solvent environment.[237,241]
Saito and co-workers reported a highly efficient reaction at 5’-ABrU-3’ sequences vs. 5’-GBrU-3’ in a series of direct oligonucleotide irradiation experiments (BrU = BrdU).[236] Further investigations carried out by the research group of Greenberg indicated that halide elimination may compete with charge migration and recombination.[242] The mechanism of degradation of BrdU-containing ODNs upon UV irradiation (302 nm) was already reported in Scheme 1.3 of Chapter 1.
The uridine-5-yl radical, generated after debromination of the photoexcited BrdU / dA couple or after single electron reduction, abstracts hydrogen from the vicinal sugar moiety, specifically from the position C1’ and/or C2’. The carbon centered radicals so formed undergo a succession of rearrangement and elimination reactions yielding alkaline labile intermediates and eventually to strand breaks (Scheme 2.9). In the presence of a hydrogen donor (H-donor) the uridine-5-yl radical eventually gives rise to the 2’-deoxyuridine (dU) after H-abstraction. The C1’ and C2’ radical chemistry has previously been described in more detail in Chapter 1.
Scheme 2.9 Pathways of degradation of BrdU-containing oligonucleotides upon single electron
injection.
All the halogenated pyrimidines are expected to exhibit higher electron affinities than their natural counterparts,[156,243] and all have been observed to capture an electron during nanosecond pulse radiolysis.[244] Specifically, the radical anion of BrdU is highly transient and decomposes with a half-life of 1.7 ns.[244] The adiabatic electron affinity was calculated using the density functional theory (DFT). For solvatated 5-Br-uracil (no sugar unit) it is in the order of 2.44 eV while 2.02 eV were determined for the natural uracil.[245] Thus, BrdU is expected to be more easily reduced than dU (or dT).
As already mentioned, BrdU was efficiently used in excess electron transfer studies by Rokita and Ito in ODNs containing a N, N, N´, N´-tetramethyl-1,5-diaminonaphtalene (TMDN) as electron donor.[33,219] The low energy excited state (λmax = 325 nm) of this strong reductant (E*ox≈ -2.8 V vs SCE) enables a selective excitation of the chromophore without direct excitation of DNA bases.[33] The reduction potential of the flavin used in the previously described EET experiments is reported to be very similar (E*ox≈ -2.6 V vs SCE).[191] All these factors persuaded to test BrdU as electron transfer acceptor in flavin-containing hairpin systems.
Electron acceptors: BrdA
The selection of 8-bromo-2’-deoxyadenosine (BrdA) as electron acceptor derives from numerous studies about this compound and the products of one-electron reduction. The above mentioned cyclonucleosides, illustrated in Chapter 1, are among these products. For example, in the case of the 5’,8-cyclo-2’-deoxyadenosine (CydA), the cyclisation was triggered by radiolytic or photolytic methods.[25,26] Specifically, reactions occurring upon single electron reduction of BrdA were investigated. Using calculations (DFT-B3LYP), pulse radiolysis and product studies, Chatgilialoglu and co-workers described kinetic and thermodynamic properties of this reaction, summarized in Scheme 2.10.[25-27,53]
Scheme 2.10 Reaction of BrdA with solvated electrons. k1 was determined by measuring the rate of the optical density decrease of e-
aq at 720 nm (ε = 1.9 x 104 M-1s-1) as a function of nucleoside concentration.[25-27,53]
Briefly, the rate constant for the reaction of solvated electrons (eaq-) with BrdA was determined by measuring the rate of the optical density decrease of eaq- at 720 nm (ε = 1.9 × 104 M-1s-1) as a function of nucleoside concentration. The bimolecular rate constant was found to be k1 = 1.6 1010 M-1 s-1. Comparing the analogous reaction with the natural adenosine (dA, k = 8.2 × 109 M-1 s-1), the presence of bromine increases the rate constant of the reduction by a factor of 2.[25]
With a rate constant of k1 = 1.6 × 1010 M-1 s-1, BrdA was chosen as fast electron sink in the following EET studies. Moreover, BrdA it is also known not to affect the stability of duplex DNA adversely,[246] whereas its use as electron acceptor in EET is unprecedented.
Electron acceptors: BrdG
8-bromo-2’-deoxyguanosine (BrdG), like BrdU and BrdA, reacts comparably fast with electrons.[247] For this reason it has been selected as electron acceptor in these EET studies as well. As already mentioned in Chapter 1, the one-electron reduction of BrdG and the ribose analogue 8-bromo-guanosine (BrG) does not follow the same pathway as BrdA. The reduced adduct undergoes protonation at C8 and tautomerization to afford the one-electron oxidised 2’-deoxyriboguanosine or riboguanosine (Scheme 2.11).[53,54,248] Nevertheless, BrdG reacts with solvated electrons very fast. A rate constant of k1 = 1.1 × 1010 M-1 s-1 was determined for the reaction of BrdG with e-aq by measuring the rate of the optical density decrease of e-aq as a function of the concentration of the nucleoside added.[53]
Scheme 2.11 Reaction of BrdG with solvated electrons. Pathway of one-electron oxidised guanosine
formation. k1 was determined as for BrdA.[54]
BrdG was also used as electron acceptor in EET investigations in G-quadruplex[249] and in double stranded DNA.[247]
The choice of BrdG as an electron acceptor in the following studies was driven by the necessity to have a larger variety of acceptors. Furthermore, the reduction of Br-purines in a duplex can yield important information for the cyclo-purine generation pathway in DNA.
In summary, the bimolecular rates of the reaction of a solvated electron with all three selected electron acceptors (BrdA, BrdG and BrdU) are known, and they are in the diffusion controlled regime[250] with about 1-2 × 1010 M-1 s-1.[25,53,251] The reduction is followed by a prompt release of Br-.[25,54,237,244] The flavin donor that will supply the electrons in the Br-nucleosides containing models has a reduction potential of E*ox≈ -2.6 V vs SCE.[191] Thus, the redox-properties of the selected Br-acceptors have to be taken into account. Although not all of the redox potential of the Br-substituted bases are known, it can be roughly assumed, based on the reduction potentials of the unmodified nucleobases, that they follow the order BrdU > BrdA > BrdG. Thus, BrdU is the easiest to reduce, followed by BrdA and BrdG. The redox potentials E° of the nucleobases are reported as T = 1.7 V, C = 1.6 V, A = 1.4 V, G = 1.3 V vs NHE,[115] whereas their rate constants (dT, dC, dA and dG) with solvated electrons are all in the diffusion controlled region of 0.6-1.8 x 1010 M-1 s-1.[252] BrdU is reported to be 40-50 mV easier to reduce than dT.[253] Thereby, BrdU, BrdA and BrdG possess the correct features for an applications as electron acceptors in combination with the flavin electron donor.